Background

Alzheimer’s disease (AD) is pathologically characterized by excessive
accumulation of amyloid-beta (Aβ) fibrils within the brain and activation of
astrocytes and microglial cells. In this study, we examined anti-inflammatory
and anti-amyloidogenic effects of 2,4-bis(p-hydroxyphenyl)-2-butenal (HPB242),
an anti-inflammatory compound produced by the tyrosine-fructose Maillard
reaction.

Methods

12-month-old Tg2576 mice were treated with HPB242 (5 mg/kg) for 1 month and
then cognitive function was assessed by the Morris water maze test and passive
avoidance test. In addition, western blot analysis, Gel electromobility shift
assay, immunostaining, immunofluorescence staining, ELISA and enzyme activity
assays were used to examine the degree of Aβ deposition in the brains of Tg2576
mice. The Morris water maze task was analyzed using two-way ANOVA with repeated
measures. Otherwise were analyzed by one-way ANOVA followed by Dunnett’s post
hoc test.

Results

Treatment of HPB242 (5 mg/kg for 1 month) significantly attenuated cognitive
impairments in Tg2576 transgenic mice. HPB242 also prevented amyloidogenesis in
Tg2576 transgenic mice brains. This can be evidenced by Aβ accumulation, BACE1,
APP and C99 expression and β-secretase activity. In addition, HPB242 suppresses
the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2
(COX-2) as well as activation of astrocytes and microglial cells. Furthermore,
activation of nuclear factor-kappaB (NF-κB) and signal transducer and activator
of transcription 1/3 (STAT1/3) in the brain was potently inhibited by
HPB242.

Conclusions

Thus, these results suggest that HPB242 might be useful to intervene in
development or progression of neurodegeneration in AD through its
anti-inflammatory and anti-amyloidogenic effects.

Alzheimer’s disease (AD) is a fatal progressive neurodegenerative illness and the
most common form of dementia [1]. AD
is a devastating dementia that first presents as progressive memory loss and later
can include neuropsychiatric symptoms [2]. Amyloidogenic processing of amyloid precursor protein (APP)
by β- and γ-secretases leads to the production of Aβ peptides that can oligomerize
and aggregate into amyloid plaques, a characteristic hallmark of AD [3]. Although the exact cause of AD remains
elusive, mounting evidence continues to support the involvement of neuroinflammation
in the development of AD [4].
Neuropathological studies in the human brain have demonstrated that the activated
glial cells excessively release pro-inflammatory mediators and cytokines, which in
turn triggerneurodegenerative cascades via neuroinflammation [5, 6]. Inflammatory reactions and mediators have been reported to
augment APP expression and Aβ formation [7, 8] and
transcriptionally upregulate mRNA and protein levels and enzymatic activity of
β-secretase, a key enzyme in the production of Aβ [9, 10].

Astrocytes and microglia are the major type of glial cells in the central nervous
system and activation of these cells are involved in all types of neurodegenerative
processes, indicating prominent remodeling in AD [11, 12].
Activated astrocytes expressing glial fibrillary acidic protein (GFAP) are closely
associated with AD pathology, such as Tau tangles, neuritic plaques and amyloid
depositions [13]. Furthermore,
astrocytes with increased beta-secretase 1 (BACE1) expression have been found in the
brain in AD [14]. Fibrillar Aβ can
activate microglia, resulting in production of toxic and inflammatory mediators like
hydrogen peroxide, nitric oxide, and cytokines [15]. Microglial cells are closely associated with nearly all
compact deposits of the Aβ-protein found in the senile plaques of AD [16]. Microglial activation is also involved
in neuroprotection in the early phase, but, subsequently, extensive and continuous
activation of microglia results in the neuroinflammation and Aβ accumulation in AD
pathology [17].

Nuclear factor-kappa B (NF-κB) is a redox transcription factor that is critical
for regulation of inflammation and various autoimmune diseases [12]. NF-κB is localized in the cytoplasm by
the inhibitor of κB (IκB). After IκB is phosphorylated and degraded, NF-κB is
released and translocated from the cytosol to the nucleus, and binds to its cognate
DNA binding sites leading to expression of inflammatory mediators [18]. Expression of genes for inflammatory
elements such as inducible inducible nitric oxide synthase (iNOS) and
cyclooxygenase-2 (COX-2), as well as cytokines, can be regulated by activation of
NF-κB [10]. NF-κB activation was
also found to promote neuronal resistance to Aβ toxicity [19]. NF-κB signaling increases BACE1
expression [20] and NF-κB
inhibitors decrease Aβ production and β-secretase activity [21]. Recently, it was reported that
inhibition of the NF-κB pathway could enhance α-secretases, but inhibit β-secretase
activity, and thereby reduce the formation of Aβ [22]. Signal transducer and activator of transcription (STAT)1 and
STAT3 are also significant regulators of neuroinflammation, Aβ generation
[23] and cytokine-driven
NF-κB-mediated Aβ gene expression [24]. It was reported that resveratrol prevented the
pro-inflammatory properties of fibrillar Aβ on macrophages by potently inhibiting
the effect of Aβ on IκB phosphorylation, STAT1 and STAT3 activation [25]. Endogenous BACE1 levels were decreased
by overexpression of suppressor of cytokine signaling (SOCS), an endogenous negative
regulator of STAT1 signaling [26],
demonstrating that downregulation of STAT1 signaling suppresses BACE1 expression and
Aβ generation in neurons [27].
STAT3 is another transcription factor that is typically associated with cytokine
signaling during neuronal differentiation and inflammation [28]. The STAT3 patwhay is disrupted in
neurodegeneration induced by amyloid-peptide [29]. The STAT3-mediated transcriptional control of BACE1 leads to
amyloidogenic processing of APP and Aβ generation [23].

Animals

In the present study we used Tg2576 mice as a model of AD. These mice
overexpress human APP with the Swedish double mutations (K670N, M671L) under the
control of a hamster prion protein promoter [33]. The 12-month-old Tg2576 female mice used in the present
study were purchased from Taconic Farms (Germantown, NY, USA) and were
maintained and handled in accordance with a protocol approved by the
Institutional Animal Care and Use Committee of Laboratory Animal Research Centre
at Chungbuk National University (Approval no. CBNU-144-1001-01).

Chemicals

Characterization of HPB242 has been described elsewhere [34]. In brief, we prepared 100 ml of
fructose-tyrosine mixture including 0.1 M tyrosine and 0.05 M fructose. MR was
carried out in temperature-controlled autoclave apparatus (Jisico, Seoul, South
Korea) at 130°C for 2 hr. After 2 hr heating, the reaction mixture was filered
through a 0.45 μm membrane and used to isolate the active compounds through
several fractionation steps. Fructose-tyrosine MR products were purified using a
series of solvent fractionations; ethyl acetate (EtOAc), n-butanol, and water.
The EtOAc fraction was subjected to silica gel column chromatography and eluted
with increasing concentrations of methanol (MeOH) in dichloromethane (DCM). An
A2 fraction via DCM:MeOH (20:1, v:v) of a first silica gel chromatography was
separated to 23 sub-fractions via a second silica gel chromatography. Fractions
B14 and B15 from the 23 sub-fractions were further purified by semi-preparative
high performance liquid chromatography with a C18 column. The structure is shown
in Figure 1A. Then
2,4-Bis(phydroxyphenyl)-2-butenal was given to Tg2576 mice (n = 8 in each group)
in drinking water at the dose of 5 mg/kg for 1 month.(Figure 1B).

Figure 1

Structure of HPB242 and the scheme of
experimental study on the mice models. A
tyrosine-fructose Maillard reaction product HPB242 (A) and scheme for animal treatments
and behavioral tests. (B) The
animals received HPB242 through drinking water (5 mg/kg) for 1
month, and then memory tests were conducted.

Morris water maze test

The Morris water maze test is a widely accepted method for examining cognitive
function and was used in the present study as described previously [35]. Briefly, a circular plastic pool
(height 35 cm, diameter 100 cm) was filled with water (plus white dye)
maintained at 22 to 25°C. An escape platform (height 14.5 cm, diameter 4.5 cm)
was submerged 0.5 to 1 cm below the surface of the water. The test was performed
three times a day for 7 days during the acquisition phase (days 1 to 7), with
three randomized starting points. The position of the escape platform was kept
constant. Each trial lasted for 60 s or ended as soon as the mice reached the
submerged platform. The swimming pattern of each mouse was monitored and
recorded by a camera mounted above the center of the pool, and the escape
latency, escape distance and swimming speed were assessed by the SMART-LD
program (Panlab, Barcelona, Spain). A quiet environment, consistent lighting,
constant water temperature and a fixed spatial frame were maintained throughout
the experimental period.

Probe test

To assess memory consolidation, a probe test was performed 48 hr after the
water maze test (on day 9). For the probe test, the platform was removed from
the pool and the mice were allowed to swim freely. The swimming pattern of each
mouse was monitored and recorded for 60s using the SMART-LD program (Panlab).
Consolidated spatial memory was estimated by the time spent in the target
quadrant area.

Passive avoidance test

The passive avoidance response was determined using step-through apparatus
(Med Associates, Georgia, VT, USA). At 48 hr after the probe test (on day 11), a
training trial was performed. To this end, each mouse was placed in the
illuminated compartment of the apparatus facing away from the dark compartment.
When the mouse moved completely into the dark compartment, it received an
electric shock (0.45 mA, 3s duration). At 24 hr after the training trial (on day
12), each mouse was placed in the illuminated compartment and the latency period
until it entered the dark compartment was determined and defined as the
step-through latency. The cut-off time for the examination was 180s.

Collection and preservation of brain tissues

At 48 hr after the passive avoidance test, mice were anesthetized with diethyl
ether and then perfused with PBS. The brains were immediately removed from
skull, and the cortex and hippocampus were dissected on ice. All brain tissues
were stored at −80°C until biochemical analysis.

Immunohistochemical staining

After being anesthetized with diethyl ether, subgroups of mice were perfused
intracardially with 50 mL saline. The brains were removed from the skull and
post-fixed in 4% paraformaldehyde for 24 h at 4°C. The brains were transferred
to 30% sucrose solutions. Subsequently, brains were cut into 30 μm sections by
using cryostat microtome (Leica CM1850; Leica Microsystems, Seoul, Korea). After
multiple washing in PBS, endogenous peroxidase activity was quenched by
incubation of the samples in 3% hydrogen peroxide in PBS for 30 minutes,
followed by a 10-minutes wash in PBS. Sections were then incubated for 2 h at
room temperature with a mouse polyclonal antibody against Aβ (1:5000; Covance,
Berkeley, CA, USA), a rabbit polyclonal antibody against GFAP and iNOS (1:1000;
Abcam, Inc, Cambridge, MA, USA), a rabbit polyclonal antibody against COX-2
(1:1000; Cayman, Ann Arbor, MI, USA) and a rabbit polyclonal antibody against
ionized calcium binding adaptor molecule 1 (Iba1) (1:1000; Wako, Osaka, Japan).
After incubation with the primary antibodies, sections were washed in PBS before
being incubated for 1 h at room temperature in the presence of biotinylated goat
anti-rabbit or anti-mouse IgG secondary antibodies (1:1000; Vector Laboratories,
Burlingame, CA, USA). Sections were then washed with PBS and incubated with
avidin-peroxidase complex (Vector Laboratories) for 30 minutes before the
immunocomplex was visualized using the chromogen 3,3′-diaminobenzidine (Vector
Laboratories). Sections were then counterstained with hematoxylin. Finally,
sections were dehydrated in ethanol, cleared in xylene and covered with Permount
(n = 8 mice per group).

Immunofluorescence staining

The brain-tissue processing methods were the same as described above
(Immunohistochemical staining). The mouse brain sections were incubated for 2 h
at room temperature with a goat polyclonal antibody against p50 (1:500, Santa
Cruz Biotechnologies, Inc., Santa Cruz, CA, USA), a rabbit monoclonal antibodies
against Iba1 (1:1000; Wako, Osaka, Japan) and a rabbit monoclonal antibodies
against GFAP (1:1000; Abcam). After washing with PBS, the brain sections were
incubated with an anti-rabbit or anti-mouse secondary antibody labeled with
Alexa-Fluor 488 and Alexa-Fluor 568 (1:800 Invitrogen, Paisley, UK) for 2 h at
room temperature. Sections were then dehydrated in ethanol, cleared in xylene
and covered with Permount. Final images were acquired using a confocal laser
scanning microscope (TCS SP2, Leica Microsystems AG, Werzlar, Germany).

Gel electromobility shift assay (EMSA)

Gel shift assays were performed according to the manufacturer’s
recommendations (Promega, Madison, WI, USA). Briefly, brain tissues (100 mg)
were suspended in 200 μl of solution A (1 M HEPES (pH 7.9), 0.15 M
MgCl2, 1 M KCl, 100 mM dithiothreitol, 0.5% Nonidet
P-40, 0.1% Protase Inhibitor (Sigma), 0.1% Phosphatase Inhibitor (Sigma) and 100
mM phenylmethylsulfonyl fluoride). Tissues were then allowed to incubate on ice
for 6 minutes and were centrifuged at 8000 rpm for 8 minutes. The pelleted
nuclei were resuspended in solution C (solution A + (5 M NaCl, 20 mM
ethylenediamine tetraacetic acid anticoagulant (EDTA), 20% glycerol)-(Nonidet
P-40, 1 M KCl)) and allowed to incubate on ice for 20 minutes. The tissues were
centrifuged at 15000 rpm for 15 minutes, and the resulting nuclear extract
supernatant was collected in a chilled Eppendorf tube. Consensus
oligonucleotides were endlabeled using T4 polynucleotide kinase and
(γ-32P) ATP for 10 minutes at 37°C. Gel shift
reactions were assembled and allowed to incubate at room temperature for 10
minutes followed by the addition of 1 μL (50,000 to 200,000 rpm) of
32P endlabeled oligonucleotide and another 20
minutes of incubation at 37°C. Subsequently 1.5 μL of gel loading buffer was
added to each reaction and loaded onto a 6% non-denaturing gel, and
electrophoresis was performed until the dye was four fifths of the way down the
gel. The gel was dried at 80°C for 2 h and exposed to film overnight at −70°C.
The relative density of the protein bands was scanned by densitometry using
MyImage (SLB, Seoul, Korea), and quantified by Labworks 4.0 software (UVP
Inc.).

Measurement of Aβ1–42

A specific ELISA kit (Immuno-Biological Laboratories, Gunma, Japan) was used
to determine Aβ(1 to 42) levels. Protein was extracted from brain tissue using
lysis buffer containing protease inhibitors and centrifuged 2500 × g for 15
minutes at 4°C. The supernatant was collected. Briefly, 100 μL aliquots of brain
tissue samples (total protein 100 μg) from eight mice in each group were added
to pre-coated plates and incubated overnight at 4°C. After washing each well
seven times with washing buffer, 100 μL chromogen-labeled antibody solution was
added and the mixture was incubated for 1 h at 4°C in the dark. After washing
each well nine times with washing buffer, 100 μL chromogen was added and the
mixture was incubated for 30 minutes at room temperature in the dark. Finally,
after added 100 μL stop solution, the resulting color was assayed at 450 nm
using a microplate reader (Sunrise; TECAN, Mannedorf, Switzerland).

Measurement of β-secretase

β-secretase activity in Tg2576 mice brains was determined using a commercially
available β-secretase activity kit (Abcam). Protein was extracted from brain
tissue using ice-cold extraction buffer, incubated on ice 20 minutes and
centrifuged 10000 × g for 5 minutes at 4°C. The supernatant was collected. A
total of 50 μL of sample (total protein 100 μg) was added to each well followed
by 50 μL of 2 × reaction buffer and 2 μL of beta-secretase substrate incubated
in the dark at 37°C for 2 hr. Fluorescence was read at excitation and emission
wavelengths of 355 and 510 nm respectively, using a Fluostar Galaxy fluorimeter
(BMG Lab Technologies, Offenburg, Germany) with Felix software (BMG Lab
Technologies, Offenburg, Germany). Beta-secretase activity is proportional to
the fluorimetric reaction, and is expressed as nmol/mg protein per
minute.

Statistical analysis

All statistical analysis was performed with GraphPad Prism 4 software (Version
4.03; GraphPad software, Inc., San Diego, CA, USA). Group differences in the
escape distance, latency, and velocity in the Morris water maze task were
analyzed using two-way analysis of variance (ANOVA) with repeated measures, the
factors being treatment and testing day. Otherwise, data were analyzed by
one-way ANOVA followed by Dunnett’s post hoc test. All values are presented as
mean ± standard error of the mean (SEM). Significance was set at P < 0.05 for all tests.

Inhibition of memory impairment in Tg2576 mice by HPB242

To investigate the preventive effect of HPB242 against memory impairment and
Aβ1-42 depositions in the AD model mice, we treated
12-month old Tg2576 transgenic mice with HPB242 for 1 month, and then compared
memory deficiency with the non-treated mice using the water maze test. The
Tg2576 mice were trained for three trials per day for 7 days. Escape latency and
escape distances, which are the time and distance travelled to reach the
platform in the water maze, were measured to determine the memory-improving
effect of HPB242. The mice exhibited shorter time and shorter escape latency
with the training, however, the escape latency of Tg2576 mice was not much
reduced compared to the non-transgenic mice. Oral treatment with HPB242 (5
mg/kg) for 1 month significantly ameliorated memory dysfunction in the AD model
mice. Statistical analysis of data from day 5 showed a significant
memory-improving effect of HPB242 treatment. Escape latency (F (1, 15) = 9.61, P < 0.05 (treatment-wise)) (F (6, 15) = 12.83, P <
0.05 (day-wise)) of the treated group was shorter than that of the non-treated
group (Figure 2A). Escape distance
(F (1, 15) = 10.31, P < 0.05 (treatment-wise); F (6, 15) = 5.51, P < 0.05 (day-wise)) was also reduced by the treatment
(Figure 2B). However, there was no
significant difference in average speed between the non-treated and the
HPB242-treated group (data not shown).

Figure 2

Effect of HPB242 on improvement of memory
impairment in Tg2576 mice. The training trial was
performed three times a day for 7 days. Swimming time (A) and swimming distance (B) to the platform were automatically
recorded. Two days after the training trials, a probe test was
performed. The time spent in the target quadrant and target site
crossing within 60 s was represented (C). Each value is presented as mean ± standard
error of the mean (SEM) from eight mice. To perform the passive
avoidance test, mice were given an electric shock on entering
the dark compartment for training on the learning day. After 2
days, the retention time in the illuminated compartment was
recorded (D). Each value is
presented as mean ± SEM from eight mice. #Significantly
different to non-Tg mice (P
< 0.05), *Significantly different to non-treated Tg2576 mice
(P <
0.05).

After the water maze test, we performed a probe test to analyze maintenance of
memory. During the probe test, the time spent in the target quadrant by the
Tg2576 mice group treated with HPB242 (18.78 ± 4.72 s) was significantly
increased compared with the non-treated group (36.87 ± 8.14 s) (F (1, 15) = 207.84, P < 0.05) (Figure 2C). In particular, the time spent by HPB242-treated Tg2576 mice
was similar to the time spent by non-transgenic mice (27.31 ± 10.73s).

We then evaluated learning and memory capacities by the passive avoidance test
using the step-through method. In the passive avoidance test, there was no
significant difference on the learning trial. However, in the test trial, Tg2576
mice treated with the HPB242 significantly increased the step-through latency
(173.33 ± 36.56 s) compared with the non-treated transgenic mice (100.16 ± 32.49
s) (F (1, 15) = 11.26, P < 0.05) (Figure 2D).

Effect of HPB242 on Aβ accumulation and amyloidogenesis in brains of Tg2576
AD mice

Several studies reported that Aβ accumulation, which is thought to be a major
cause of AD, occurred in the brain of Tg2576 mice. Hence, we investigated
whether HPB242 attenuated Aβ accumulation in the brains of Tg2576 mice.
Immunohistochemical analyses using Aβ1–42-specific
antibodies revealed clearly the Aβ deposition in the cortex and hippocampus of
non-treated Tg2576 mice. In contrast, there appeared to be reduced Aβ
accumulation in the brains of HPB242-treated Tg2576 mice, as evidenced by a
decrease in the number of Aβ plaques (Figure 3A). To determine whether the degree of Aβ deposition is
paralleled with Aβ protein levels in the brain, quantitative analysis of
Aβ1–42 levels was performed using an
Aβ1–42-specific ELISA kit. As shown in Figure
3B, HPB242 treatment reduced
Aβ1–42 levels in the brain of Tg2576 mice. In
addition, to evaluate β-secretase activity (BACE1) we performed ELISA, and
western blot analysis was performed to quantify β-secretase activity, APP and
BACE1. ELISA analysis revealed that β-secretase activity was significantly
reduced by treatment with HPB242 in the brain of Tg2576 mice (Figure
3C). Western blot analysis also
revealed that APP, C99 and BACE1 levels were significantly decreased in both the
cortex and hippocampus of HPB242-treated mice compared with non-treated mice
(Figure 3D).

Figure 3

Inhibitory effects of HPB242 on
accumulation of Aβ1-42in the brain of Tg2576 mice. Aβ
accumulation in the brains of Tg2576 mice was determined by
immunohistochemical analysis using
Aβ1-42-specific antibody (A). The sections of Tg2576 mouse
brains were incubated with anti-Aβ1-42
primary antibody, and biotinylated secondary antibody.
Immunoperoxidase staining of brains of Tg2576 and treated-Tg2576
mice shown (brown color). Aβ1-42 level
was measured in mouse brains by ELISA as described in Materials
and Methods (B). The value is
mean ± standard error of the mean (SEM) (n = 8 mice). The
activity of β-secretase was investigated using assay kit as
described (C). Values measured
from each group of mice were calibrated by the amount of protein
and expressed as mean ± SEM (n = 8 mice). The expression of APP
and BACE1 were detected by western blotting using specific
antibodies in the mouse brain. Each blot is representative of
three experiments (D).
*Significantly different from non-treated Tg2576 mice (P < 0.05).

Effect of HPB242 on activation of astrocytes and microglia, and expression
of iNOS and COX-2 in the Tg2576 mice brain

It has also been reported that activation of astrocytes and microglia is one
of the characteristic features of AD; these can produce pro-inflammatory
cytokines as well as generate Aβ on activation. We thus examined activation of
astrocytes and microglia in the brains of HPB242-treated and non-treated Tg2576
mice. In the results, GFAP-reactive cell number (activated astrocytes) and
Iba1-reactive cell number (microglia) was reduced in the brains of
HPB242-treated Tg2576 mice compared to those of non-treated Tg2576 mice (Figure
4A and B). To confirm these results,
we investigated the expression of GFAP and Iba1 by western blot analysis. The
results revealed that GFAP and Iba1 levels were significantly decreased in both
the cortex and hippocampus of HPB242-treated compared with non-treated mice
(Figure 4C). We also investigated the
inhibitory effect of HPB242 on memory impairment and Aβ deposition via
inhibition of neuroinflammation; the expression of iNOS and COX-2 were
determined by immunohistochemical analysis and western blot. Expression of the
inflammatory protein such as iNOS and COX-2 in brain of Tg2576 mice were
significantly decreased by treatment of HPB242 (Figure 4D, E and F).

Figure 4

HPB242 inhibited activation of astrocytes
and microglia, and reduced expression of COX-2 and iNOS in
the brains of Tg2576 mice. The effect of HPB242
on reactive astrocytes and activated microglia cells was
measured by immunohistochemical analysis and western blotting
analysis. The sections of mice brain incubated with anti-glial
fibrillary acidic protein (GFAP) (A) or ionized calcium binding adaptor molecule 1
(Iba1) (B) primary antibody and
the biotinylated secondary antibody (n = 8). The stained tissues
were viewed with a microscope (×100 or 400). Expression of GFAP
and Iba1 were also examined by specific antibodies in the cortex
and hippocampus (C). Each blot
is representative for three experiments (n = 8). Inhibitory
effects of HPB242 on the Tg2576 mice brain expression of
inflammatory proteins. The sections of mouse brain incubated
with anti-iNOS (D) or COX-2
(E) and the biotinylated
secondary antibody (n=8). The resulting tissue was viewed with a
microscope (×100 or 400). The expression of inducible nitric
oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected
by western blotting using specific antibodies (F). Each blot is representative for
three experiments (n = 8). *Significantly different to
non-treated Tg2576 mice (P
< 0.05).

Effect of HPB242 on NF-κB transcriptional and DNA binding in the brain of
Tg2576 mice

Since NF-κB is implicated for Aβ generation and neuroinflammatory reaction, we
determined NF-κB DNA binding activity and expression of p50 and p65, submits of
NF-κB. The analysis of NF-κB translocation on the Tg2576 mouse brain was
performed by confocal microscopy. From confocal microscopy analysis, we found
that HPB242 prevented NF-κB translocation from cytoplasm to nucleus in the
microglia and astrocytes of the Tg2576 mouse brain (Figure 5A and B). Moreover, to confirm this detail, we
also investigated the effects of HPB242 on NF-κB activation in Tg2576 mice. This
DNAbinding activity of NF-κB confirmed by EMSA analysis was inhibited by HPB242
(Figure 5C). The levels of cytoplasmic
phosphorylated IκB, nuclear translocation of p50 and p65 were also inhibited by
treatment with HPB242 (Figure 5D).

Figure 5

Inhibitory effect of HPB242 on nuclear
factor-kappa B (NF-κB) translocation and DNA binding
activity in the Tg2576 mouse brain. Effect of
HPB242 on translocation of p50 in the Tg2576 mice brain;
confocal microscopy images of the nuclear translocation of
NF-kB-p50 were observed in microglia (A) and astrocytes (B) in the Tg2576 mouse brain. Effect of HPB242
on NF-κB activity in the Tg2576 mouse brain were determined by
gel electromobility shift assay (EMSA) as described in Materials
and Methods (C). Representative
results were obtained from at least three different sets of
experiment (n = 8). Inhibitory effect of thiacremonone on
nuclear translocation of p50 and p65 and effect of HPB242 on
p-IκB and IkB expression in the Tg2576 mice brain (D). Nuclear translocation of p50,
p65, p-IκB and IκB expression levels in activity in the Tg2576
mice brain were determined by western blot (n = 8).

Effect of HPB242 on STAT1 and STAT3 activities

In the nucleus, STATs and NF-κB regulate the activity of genes whose products
are critical in controlling numerous cellular and organismal processes,
including inflammatory responses and Aβ deposition in the brain in. We
investigated whether HPB242 can prevent activation of STAT1 and STAT3 in the
brain of Tg2576 mice. Our results showed that the DNA bingding ability of STAT1
and STAT3 was inhibited by HPB242 performed by EMSA (Figure 6A). Western blot analysis revealed that p-STAT1
and p-STAT3 levels were significantly decreased in the whole brain of
HPB242-treated Tg2576 mice compared with non-treated Tg2576 mice (Figure
6B).

Figure 6

Effects of HPB242 on signal transducer and
activator of transcription (STAT)1 and STAT3 activation in
the Tg2576 mice. Gel electromobility shift assay
(EMSA) analysis of STAT1 and STAT3 DNA binding activity in
nuclear extracts from the brains (cortex and hippocampus) of
HPB242-treated and non-treated Tg2576 mice(A). The retarded bands are indicated by an
arrow. Representative results were obtained from at least three
different sets of experiment (n = 8). The Tg2576 mouse brain
extracts were prepared, and phospho-STAT1 and phospho-STAT3
level were detected by western blot (B). Each blot was representative of three
experiments (n = 8).

Accumulating epidemiological evidence has suggested that neuroinflammation may
contribute to the occurrence and progression of AD [36–38]. The brains of patients with AD appear to display enhancement
of hallmarks of neuroinflammation, including marked astrogliosis, and elevated
release of proinflammatory mediators and cytokines, as well as microglial activation
[6]. Indeed, neuroinflammation
has been reported to cause amyloidogenesis in several AD animal models. For example,
APP/PS1 is an AD mouse model bearing mutant transgenes of APP and PS1; Aβ deposition
and neuroinflammation were present in this mouse model in the early stage of life
[39, 40]. We also reported that Tg2576 mice
displayed Aβ deposition, neuronal dysfunction, neuroinflammation and impairment of
spatial memory by overexpression of APP [41]. Furthermore, neuroinflammatory reaction has been detected in
Aβ-infused mice [42, 43]. It was also reported that several
anti-inflammatory compounds such as 4-O-methylhonokiol, thiacremonone and obovatol
improved memory functions in AD animal models [6, 12,
42]. In the present study
HPB242 inhibited memory impairment, and suppressed amyloidogenesis via its
anti-neuroinflammatory properties in Tg2576 mice. These results indicated that
anti-neuroinflammatory effects of HPB242 could be associated with
anti-amyloidgenesis, and thus improve memory dysfunction.

BACE1 cleaves APP at the N-terminal position of Aβ [44]. It was reported that the Swedish mutant
Tg2576 mice show enhanced cleavage of APP by increase of β-secretase activation
[45]. It is known that Aβ is
generated from APP by a series of proteolytic processes involving β- and
γ-secretases in the amyloidogenic pathway [46]. In the present study, we found that HPB242 reduced Aβ
accumulation in Tg2576 mice through inhibition of β-secretase activity and
consecutive decrease in expression of APP and C99. Recently, it was also reported
that the anti-inflammatory compound, thiacremonone, a sulfur compound isolated from
garlic, and 4-O-methylhonokiol effectively inhibited amyloidogenesis through reduced
β-secretase activity in LPS-injected mice [12, 47].
Activated astrocytes and microglia closely associate to amyloid plaques in AD. They
could have a role in the neurotoxicity observed in AD because of the inflammatory
reaction they generate [48]. BACE1
is induced in the proximity of activated astrocytes and microglia and upregulated in
astrocytes and microglia upon exposure to pro-inflammatory cytokines influencing APP
processing and β-secretase activity, to induce amyloidogenesis [49, 50]. We found that the number of reactive astrocytes and
microglia were elevated in the brain of Tg2576 mice, which was prevented by HPB242.
HPB242 also demonstrated anti-inflammatory and anti-amyloidogenic effects in the
Tg2576 mouse brain. Similar to these in vitro
effects of HPB242-like anti-inflammatory and anti-amyloidogenic effects
[10], it was also reported that
BACE1 transcription is increased by other inflammatory gene expression such as iNOS
and COX-2, which are regulated by NF-κB in reactive astrocytes and microglia
[51]. In this study, we also
found that HPB242 inhibits expression iNOS and COX-2, especially in the hippocampus
of Tg2576 mice. Moreover, we found that the reactive cell number for GFAP and Iba1
are also reactive for p50, and the elevated co-reactive cell numbers in the Tg2576
mouse brain was reduced by HPB242. These results suggest that the inhibitory effect
of HPB242 on the activation of astrocytes and microglia might be significant to
modulate or halt neuroinflammatory-mediated amyloidogenesis.

NF-κB is a positive regulator in the expression of a variety of rapid-response
genes involved in inflammatory effects and amyloidogenesis [52]. NF-κB activates the transcription of
APP, BACE1 and some of the γ-secretase members and increases protein expressions and
enzymatic activities, resulting in enhanced Aβ production [53]. We therefore investigated the activity
of NF-κB as a possible mechanism of the anti-inflammatory and anti-amyloidogenesis
effect of HPB242. In this study we showed that HPB242 reduced NF-κB activity in the
Tg2576 mouse brain. In fact, in the previous in
vitro studies, we found that HPB242 prevented LPS-induced
neuroinflammation and amyloidogenesis in cultured astrocytes and microglia through
inactivation of NF-κB [10]. In
addition, numerous factors were reported to inhibit amyloidogenesis via suppression
of NF-κB such as sorafenib [54],
L-theanine [55] and tripchlorolide
[56]. Further support comes
from a previous study showing that in AD the brain contains increased levels of
BACE1, C99 and NF-κB, and NF-κB expression leads to increase of BACE1 promoter
activity and BACE1 transcription, while knockout of NF-κB decreases BACE1 gene
expression in LPS-injected mice [6].
Moreover, in the present study, we also found that p50 and p65 subunits of NF-κB are
translocated into the nucleus of astrocytes and microglia in the brains of Tg2576
mice, and then the translocation of p50 and p65 are inhibited by the treatment of
HPB242. It was reported that Aβ activated the NF-κB pathway by selectively inducing
the nuclear translocation of the p50 and p65 subunits, and promoted an apoptotic
profile of gene expression [19].
Consistent with the effects on amyloidogenesis as well as C99 and BACE1 expression,
NF-κB-induced increase in β-secretase activity was also prevented by HPB242.
Therefore, inhibiting the effect of NF-κB could be significant in the
anti-inflammatory and anti-amyloidogenic effect of HPB242.

STAT1/3 may also be activated in glial cells by a number of cytokines and then
translocated from the cytosol to the nucleus [57]. In the nucleus, STATs regulate the activity of genes whose
products are critical in controlling β-secretase activity [58]. STAT1/3 is transcriptionally activated
by binding to the STAT1/3 binding sequence in the BACE1 promoter region; it is
interesting to note that a number of transcription factor binding sites become
activated in response to Aβ generation [45]. It has been reported that a humanin (HN) derivative named
colivelin completely restored cognitive function in Tg2576 mice by activating the
STAT3 [29]. In this study, we
showed that HPB242 inhibited phosphorylation of STAT1/3 as well as DNA binding
activity. In fact, in the previous studies we also found that blocking STAT3
abolished the inhibitory effect of HPB242 on NF-κB and amyloidogenesis induced by
LPS [10]. Additionally, endogenous
BACE1 levels were decreased by overexpression of SOCS, an endogenous negative
regulator of STAT1 signaling [26],
demonstrating that downregulation of STAT1 signaling suppresses BACE1 expression and
Aβ generation in neurons [27].
These findings suggest that one mechanism by which HPB242 prevents
anti-amyloidogenesis is due to a decrease in phosphorylation of STAT1 and STAT3. In
conclusion, our data showed that HPB242 can protect Tg2576 mice from memory
impairment through inhibition of NF-κB and STAT1/3, which could result in the
inhibition of Aβ1-42 accumulation by attenuating β-secretase
activity. We suggest that HPB242, a new product from a tyrosine-fructose MR, could
be useful for treatment and/or prevention of neuroinflammatory diseases such as
AD.

Acknowledgements

This work was supported by a grant from the National Research Foundation of
Korea (NRF) funded by the Korean Government (MEST; MRC, 2011–0029480), by a grant
(A101836) from the Korean Health Technology R&D Project, Ministry for Health,
Welfare and Family Affairs, Republic of Korea and by the Priority Research Centers
Program through the NRF funded by the Ministry of Education, Science and Technology
(2011–0031403).

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

J-TH designed the study and prepared the manuscript. PJ and J-A K performed
experiments. H-SJ isolated and characterized 2,4-bis(p-hydroxyphenyl)-2-butenal.
D-YC and Y-JL discussed the study. All authors have read and approved the final
version of this manuscript.

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